Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons

• Published in: Proceedings of the National Academy of Sciences - PNAS 2017-03, Vol.114 (12), p.E2466-E2475
• Main Authors: Gopal, Pallavi P., Nirschl, Jeffrey J., Klinman, Eva, Holzbaur, Erika L. F.
• Format: Article
• Language: English
• Subjects: Amyotrophic lateral sclerosis; Amyotrophic Lateral Sclerosis - genetics; Amyotrophic Lateral Sclerosis - metabolism; Animals; Axons - chemistry; Axons - metabolism; Biological Sciences; Cells; Cells, Cultured; Cytoplasmic Granules - chemistry; Cytoplasmic Granules - genetics; Cytoplasmic Granules - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Humans; Motor Neurons - chemistry; Motor Neurons - metabolism; Mutation; Neurons; PNAS Plus; Proteins; Rats; Rats, Sprague-Dawley; Ribonucleic acid; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; RNA; Viscosity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1614462114

Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid–liquid phase separation and liquid-like non–membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.